Continuous protein refolding in a tubular reactor

Continuous protein refolding by direct dilution was established in a laboratory-scale tubular reactor. Advanced refolding strategies by pulsed refolding and temperature leap, favoring the native pathway of specific model proteins were also implemented. The reactor was tested with two autoprotease fusion proteins, EDDIE-pep6His and 6His-EDDIE-GFPmut3.1, and also on carbonic anhydrase II. Direct refolding kinetics approximated by a folding kinetic of first order and aggregation kinetic of second order was similar to batch processes, while pulse and temperature leap refolding yields were higher than direct batch refolding method. Productivity comparisons calculated as amount of refolded protein per reactor volume and process time showed that an optimal refolding concentration to achieve highest productivity value is a balance between a rational reactor volume and a reasonable refolding time. Productivity in a tubular reactor is always higher as emptying and refilling times required for batch reactor decreases productivity. This productivity improvement is higher for a fast refolding protein than a slower one.